Nucleic Acids Research, Vol 26, Issue 10 2366-2373, Copyright © 1998 by Oxford University Press
R Meima, GJ Haan, G Venema, S Bron and S de Jong
Previous work in our group indicated that structural plasmid instability in
Bacillus subtilis is often caused by illegitimate recombination between
non-repeated sequences, characterized by a relatively high AT content.
Recently we developed a positive selection vector for analysis of plasmid
recombination events in B. subtilis which enables measurement of
recombination frequencies without interference of selective growth
differences of cells carrying wild- type or deleted plasmids. Here we have
used this system to further analyse the sequence specificity of
illegitimate plasmid recombination events and to assess the role of the
host-encoded DNA topoisomerase I enzyme in this process. Several lines of
evidence suggest that single- strand DNA nicks introduced by DNA
topoisomerase I are a major source of plasmid deletions in pGP100. First,
strains overproducing DNA topoisomerase I showed increased levels of
plasmid deletion. Second, these deletions occurred predominantly (>90%
of the recombinants) between non-repeated DNA sequences, the majority of
which resemble potential DNA topoisomerase I target sites. Sequence
alignment of 66 deletion end-points confirmed the previously reported high
AT content and, most importantly, revealed a highly conserved C residue at
position -4 relative to the site of cleavage at both deletion termini.
Based on these genetic data we propose the following putative consensus
cleavage site for DNA topoisomerase I of B.subtilis: 5'-
A/TCATA/TTAA/TA/TA-3'.
ARTICLES
Sequence specificity of illegitimate plasmid recombination in Bacillus subtilis: possible recognition sites for DNA topoisomerase I
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
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